The Gram stain is universally used in microbiology as the initial step in identifying bacteria. It involves the use of crystal violet and iodine to stain fixed bacterial cells. Gram positive bacteria can be distinguished from Gram negative bacteria by their ability to retain the purple color of the crystal violet stain after washing with alcohol or acetone. Gram negative bacteria lose the purple color during the wash and are stained pink by the counter-stain, usually safranin or basic fuchsin, applied after the wash. Since Hans Christian Gram published his method for staining bacteria in 1884, demarcation of bacteria into so-called “Gram positive” and “Gram negative” types has become the first step in classifying and subdividing bacteria.
Since the Gram reactions are based on the physical properties of the cell wall they are inherently variable. Phenotypic properties such as presence of capsules and cell age as well as external factors such as growth conditions, and anti-bacterial agents affect the cell wall and can alter the staining qualities of the bacteria. The quality of a Gram stain can be affected by external factors as well. Gram staining is easily affected by technique—particularly the decolorization step (washing) of the assay. The percentage of acetone in the decolorizer, method of application, the thickness of the specimen, and the type of specimen can all effect the exposure time required for proper decolorization. If a preparation is over-decolorized, Gram types can be confused, also crystal violet precipitates can be mistaken for bacteria.
Not all bacteria are amenable to Gram staining. Some species are Gram-variable, taking on both Gram-positive and Gram-negative colorations. It is estimated that up to 10% of positive blood cultures yield Gram-variable bacteria. Other species react weakly with the Gram stain, making them Gram-indeterminate.
In the US, there are approximately 30 million blood cultures processed annually. The average positivity rate of these blood cultures is 10%, resulting in ˜3 million positive blood cultures per year. It is a universal practice to test a positive blood culture to determine its Gram-stain result and thereby it's clinical/therapy implication for first line therapy choice. According to a recent study (Rand et al. Am. J. Clin. Pathol. 2006; 126:686-690), a retrospective, 23 month review of over 8,000 Gram stains revealed an error rate of ˜0.7%. Extrapolation of this error rate nationwide would result in 21,000 Gram stain errors per year. Laboratories may also struggle with the differentiation of cocci in clusters vs. pairs and chains or for Gram-variable bacteria.
Improvements to the basic Gram stain technique have occurred. Automation of the Gram stain technique has been implemented to lower the variability from person to person and day to day. Alternate methods have been developed which effectively separate bacteria in to Gram types. Alternative dyes and methods have been developed to simplify the use of effective gram staining in specialized scenarios (Yazdankhah et al).
Sizemore et al describe a method employing fluorescein labeled wheat germ agglutinin to selectively label gram-negative bacteria. The method suffers the same downfalls of the original method in that it is susceptible to the inherent biological variability of the Gram stain target; namely the cell wall of the organisms and it lacks a way of positively identifying gram-positive organisms. Mason et al, and Holm and Jespersen made improvements to this method, and applied it to flow cytometric analysis.
U.S. Pat. Nos. 4,639,421 and 4,665,024 describe methods of differential staining of Gram types by application of two fluorescent dyes, in general a generic dye which is taken up by all organisms, and a second dye which is preferentially taken up by gram-negative organisms. The ratio of intensity of the dyes compared to a control is used to assign Gram-type.
Bidnenko et al describe a method of differentiating Gram types via differential penetration of high molecular weight peroxidase labeled DNA probes directed at cellular ribosomal RNA targets. Penetration of the peroxidase labeled probe into the cell indicate a Gram-negative organism since Gram-positives are impenetrable to high molecular weight molecules. Combined with a second probe of a different color which non-specifically labeled bacteria the presence of a fluorescent signature of the first label or coincidental detection of the first and second label, the organisms could be typed. This method uses the inherently variable cell well as a method to differentiate bacteria into Gram types, although the method overcame the lack of positive identification of gram-positive organisms which plagued the Sizemore method. It is important to point out that the Bidnenko method used a “universal” probe for detection of all bacteria. The method, again, relied on the different properties of the outer membrane of gram negative and gram positive organisms, not on the specificity of the probe to sort microorganisms into different gram types.
As is described in U.S. Patent Publication No. 2002/0081606, 2004/0171007, 2008/0118923 and elsewhere, several groups have used the polymerase chain reaction (PCR) as a way to amplify specific nucleic acid targets indicative of either Gram-positive or Gram-negative organisms (also see Carroll et al 2000, Klashick et al 2002). 16S rRNA (US2008/0118923), 23S rRNA (US 2004/0171007) or other genes such as sodA (US 2002/0081606) are used as amplification targets and Gram type is determined by signature of the amplicon. These methods although powerful are prone to the downfalls of amplified assays including false positives cause by target contamination, loss of distinction in mixed cultures, and destruction of the organism of interest, such that morphology information is not provided.
Hybridization assays directed at rRNA targets are frequently used to differentiate bacteria by species, genus, family, etc. Jansen, et al describe a method employing probe cocktails to detect virtually all potential organisms in a sample, and differentiating them into classes. The same basic method is used broadly. Roller et al describe a similar hybridization method to detect and differentiate most high G+C content gram-positive organisms. The authors suggest the method provides information relevant to monitoring sample populations in environments where obtaining classical staining information is unreliable.
The use of Gram stain to categorize bacteria has considerable problems related to the variability of the method from person to person, the inherently variable biological properties of the organisms, the subtleness and ability to accurately detect the indicating color change, and the presence of organisms which do not fit conveniently into either type (Gram variable or Gram intermediate).
Methods are needed which do not rely on the phenotypic characteristics such as membrane composition, have clear high contrast signals, use standard microbiological tools and techniques, and do not produce variable or indeterminate results.